Pulmonary surfactant is a phospholipid-rich mixture which is secreted into the alveolar space by the type II epithelial cell. Formation of a surfactant film at the epithelial-air interface helps to maintain alveolar integrity during respiration by reducing surface tension along the alveolar epithelium. The adsorption, spreading and surface tension-reducing properties of the surfactant phospholipid film are facilitated by specific surfactant-associated proteins, such as SP-B.
Respiratory Distress Syndrome (RDS) is a major cause of mortality among prematurely born infants. It is believed that RDS is caused by a deficiency of lung surfactant. RDS can be effectively treated by administering surfactant proteins and lipids to those individuals suffering from RDS. Purified preparations of surfactant proteins, particularly surfactant protein-B (SP-B) and surfactant protein-C (SP-C), enhance the rate of formation of a phospholipid film at the air-liquid interface, and thus, promote gas exchange in those having RDS. Several lines of evidence indicate that SP-B is a critical component of surfactant: 1) Surfactant replacement preparations containing SP-B as the only protein component were equivalent to natural surfactant and were superior to comparable preparations containing surfactant protein-A SP-A and/or SP-C when tested in vivo (Am. Rev. Resp. Dis. 147:669-676, 1993). 2) Addition of SP-B to currently used replacement surfactant preparations (such as Survanta) results in improved surfactant function when tested in vivo (Pediatr. Res. 37:271-276, 1995). 3) Mutations in the SP-B gene leading to absence of SP-B in surfactant invariably result in respiratory distress syndrome and death shortly after birth (N. Engl. J. Med. 328:406-410, 1993; Proc. Natl. Acad. Sci. U.S.A. 92:7794-7798, 1995). 4) Inactivation of SP-B in the airway by interaction with antibodies specific for SP-B leads to respiratory distress syndrome and death (Exp. Lung Res. 14:247-260, 1988; J. Appl. Physiol. 71:530-536, 1991).
Human SP-B is synthesized by the type II epithelial cell as a 381 amino acid preproprotein (as shown in sequence I.D. No. 1). The 79 residue mature SP-B peptide SEQ ID NO: 2 is extremely hydrophobic and flanked by propeptides of 200 and 102 amino acids at its NH.sub.2 - and COOH-terminus, respectively SEQ ID NO: 1.
Processing of the precursor protein within the secretory pathway results in cleavage of the signal peptide, followed by proteolytic cleavage of the NH.sub.2 -terminal and the COOH-terminal propeptides SEQ ID NO: 1 to generate the biophysically active, mature peptide SEQ ID NO: 2. Proteolytic processing of the proprotein occurs in an endosomal/lysosomal compartment, the multi-vesicular body, prior to incorporation of the mature peptide into the lamellar body, which is secreted in response to an appropriate extra cellular stimulus.
The mature SP-B peptide is very hydrophobic, as it consists of approximately 60 percent non-polar amino acids SEQ ID NO: 2. In addition, SP-B is positively charged and has been shown to promote aggregation, disruption and fusion of liposomes containing negatively charged phospholipids. The potential of SP-B to disrupt lipid membranes indicates that the mature peptide must be escorted through the secretory pathway prior to association with surfactant phospholipids.
Pilot-Matias et al, DNA (1989), Vol. 8, No. 2 discloses the isolation, characterization, sequence, and chromosomal localization of the gene encoding human SP-B, as well as the complete nucleotide sequence and deduced amino acid sequence for SP-B. See also Schilling et al, U.S. Pat. No. 4,933,280. The production of mature SP-B through recombinant DNA technology is disclosed in Yao et al, Biochem Cell Biology (1990), Vol. 68:559 and Shilling et al, WO88/05820. Various references disclose the expression of surfactant proteins in a variety of systems including the use of fusion proteins in bacterial systems. Schilling et al, WO88/05820; Shilling et al, U.S. Pat. No. 5,430,020.
While the literature discloses the isolation, characterization, sequence, chromosomal localization, and production of SP-B proprotein through recombinant DNA technology, and the isolation and purification of various proteins including SP-B, the literature does not disclose an efficient process for synthesizing mature SP-B recombinantly both in vivo and in vitro, and isolating and purifying the same in a simple, rapid and economically inexpensive process. The prior art discloses methods of recombinantly producing SP-B that require numerous intricate steps of expression, isolation, purification and cleavage which result in very poor yields of mature SP-B. The purification step alone may involve various extracts and solvents which is inefficient, time consuming, and expensive.